Main Conference - Day 3 (May 22)Thursday, 22 May 2025

sgRNA sequence length and structure provide extra analytical challenges compared to siRNAs and ASOs. However, limited work to enable high resolution separations of sgRNAs have been reported. Here, we perform a systematic evaluation of chromatographic parameters to develop a highly selective IP-RPLC separation for sgRNAs, enabling single nucleotide resolution.

The fast development of therapeutic mRNAs has somewhat outpaced the analytical characterization. Challenges are associated with the verification of the sequence, labor-intensive determination of the polyA tail length and capping efficiency, and identification of impurities. A unified platform is presented for the online determination of multiple mRNA quality attributes.

We will present development of analytical methods for impurity profiling of siRNAs. Optimization of chromatography resolution and differentiation of co-eluting impurities by MS will be covered. Factors affecting the stability of double-strand oligonucleotides will be discussed as well.

Amgen Speaker TBA

Determining chiral purity is critical to evaluating the quality of peptide pharmaceutical products. For synthetic peptides, undesirable D-isomers can be introduced as impurities throughout the process. We developed an HPLC-MS/MS method for determining D-isomer impurities in synthetic peptides that is simplified and more robust compared to existing enantiomeric purity methods.

Peptide aggregation remains a significant challenge in pharmaceutical development impacting solubility, stability and therapeutic efficacy of peptide-based drugs. It can be driven by numerous factors and conditions complicating the manufacturing and storage of those molecules. This presentation explores the current understanding of peptide aggregation mechanisms. Key challenges in identifying and mitigating aggregation during manufacturing processes will be discussed. Case studies on complex long peptide sequences prone to aggregation will be shared.

Wave’s AIMers are short, chemically modified oligonucleotides that direct A-to-I RNA editing via endogenous ADAR enzymes. We will describe AIMer design optimization, which enhances editing potency, supports editing across the CNS of nonhuman primates, and enables protein restoration in a mouse model for Rett Syndrome (RTT), a genetic neurological disorder.

Prime Editors can correct almost all types of disease causing mutations with precision, high efficiency and without double strand breaks. Prime has developed a Universal LNP for liver delivery of Prime Editors. Our initial focus is on Wilson’s disease and Glycogen Storage Disease type I. We will review the steps in developing RNA-LNP Prime Editors for therapeutic use.

Changes to an established synthesis of an oligonucleotide can alter the diastereomer profile. Understanding those differences is not only a regulatory expectation but supports program strategies for clinical trials, scale-up and long-term manufacturing. Here we described orthogonal methods and techniques to evaluate these profiles on a fully thiolated ASO.

Disulfide constrained peptides (DCPs) have gained increased attention as a drug modality due to their exceptional stability and combined advantages of large biologics and small molecules. Chemical synthesis, although widely used to produce DCPs, is associated with high cost both economically and environmentally. To reduce the dependence on solid phase peptide synthesis and the negative environmental footprint associated with it, we present a highly versatile, cost and environmentally friendly bioproduction platform to generate DCPs and their conjugates, as well as chemically modified or isotope labeled DCPs. Using the DCP against the E3 ubiquitin ligase ZNRF3, MK1-3.6.10, as a model peptide, we have demonstrated the use of bacterial expression, combined with Ser ligation, to produce multivalent MK1-3.6.10 and MK1-3.6.10 with N-terminal functional groups. We have also developed a bioproduction method for site-specific incorporation of unnatural amino acids into recombinant DCPs by the amber codon suppression system. Lastly, we produced 15N/13C-labeled MK1-3.6.10 with high yield and assessed the performance of a semi-automated resonance assignment workflow that could be used to accelerate binding studies and structural characterization of DCPs. This study provides a proof of concept to generate functionalized DCPs using bioproduction, providing a potential solution to alleviate the reliance on hazardous chemicals, reduce the cost and expedite the timeline for DCP discovery.

Merck Speaker TBA

This presentation will discuss: 1) An in vivo–based genome editing approach using the clinically validated HBG1/2 promoter–targeted gene editing strategy offers potential to significantly decrease the burden for patients living with β-hemoglobinopathies and provides an opportunity for global access and 2) The optimization and evaluation of LNP formulations that deliver AsCas12a mRNA, plus HBG1/2 guide RNA, to HSPCs in the bone marrow of humanized NBSGW mice to induce fetal hemoglobin.

The current process of oligonucleotide manufacturing is time-consuming, costly, and material-intensive, often encountering significant process limitations. To enhance manufacturing efficiency, recent research has concentrated on fluidized bed solid phase synthesis and membrane-driven liquid phase synthesis. Our objective is to present a different vision for the efficient, continuous synthesis of oligonucleotides through SPS.

We developed a new ASO preparation platform, the ASO Hybrid Synthesis Strategy. With this approach, protected oligonucleotide fragments are prepared through solid-phase synthesis and then assembled using a liquid-phase process. The Hybrid Oligonucleotide Synthesis Strategy reduces LPOS production cycle time and facilitates the use of LPOS material to support early-stage clinical studies.

The solid phase oligonucleotide synthesis based on sequential coupling of phosphoramidite monomers is a well-established industrial manufacturing process currently performed at kilo scale. However, inherent limitations of synthesis and purification prevent scale-up to the high-volume needs of future siRNA therapeutics, and novel approaches need to be designed and developed to support their commercialization. Alternative synthesis and purification technologies towards more efficient, scalable, and sustainable large-scale manufacture will be discussed.

Kailera is a clinical-stage company advancing a broad, differentiated and clinically validated portfolio for the treatment of obesity, with oral and injectable programs leveraging several key mechanisms of actions and routes of administration. Our lead program, KAI-9531, a dual GLP-1/GIP receptor dual agonist, demonstrated compelling results in Phase-2 trials in China, thus we are preparing for Global US phase-3 program.

Tune-401 is a first-in-class epigenetic silencer designed to treat chronic hepatitis B (CHB). TEMPOs mechanism of action targets and silences the expression of the viral genome in infected liver cells, reducing the virus's ability to replicate and persist. This novel approach offers a promising therapeutic strategy for CHB by addressing the underlying epigenetic mechanisms that contribute to chronic infection, potentially leading to a functional cure for patients.

Editas achieved a high level of editing, protein knockdown, and biomarker reduction in non-human primate for 2 different liver targets and using 2 different LNPs, and demonstrates the first in vivo proof of concept using AsCas12a Editas is building potency correlations between in vitro tools and preclinical animal models (mouse and NHP) to enable pipeline development.

A new SPOS fluid bed synthesizer was created that improves scalability, purity, and yield compared to traditional packed bed synthesizers, while also reducing solvent and waste. siRNA API was produced in GMP manufacturing with 9% higher SS purity than previous GMP batches and 3X less ACN wash solvent.

Oligonucleotide purification traditionally has relied on a batch chromatography (AEX, HIC or HPLC) for purification, often using linear gradients w/fractionation. However, to maximize productivity, we aim here to intensify a process to meet throughput demands without giving up purity. This talk will describe how we improved throughput via going “continuous”.

Oramed's novel POD^TM technology enables seamless formulation of macromolecules for oral delivery by providing a protective milieu and promoting peptide absorption across the intestinal epithelium. Its efficacy has been demonstrated with insulin, coronavirus vaccine peptides, GLP-1, leptin and others. The platform promises to revolutionize drug bioavailability and patient care.

Effective oral delivery of biologics is a big challenge. Biograil has developed the oral capsule technology, BIONDD™, which delivers similar exposure as injections of various biomacromolecular drugs. The technology does not include enhancers or chemicals to facilitate absorption but a simple physical mechanism to achieve delivery across the mucosal barrier.

Monthly dosing of anti-obesity peptides is a highly coveted but lofty objective. Currently, most anti-obesity peptides have half-lives of one or two weeks. Monthly dosing intervals can be achieved with increased dosing, but only at the risk of adverse effects from high C_max. Here, we describe a technology that easily achieves the sought-after monthly dosing with low C_max and should even extend the dosing interval to three months or longer.

Intellia is a leading clinical-stage gene editing company focused on the development of CRISPR-based therapies. Interim clinical data with nexiguran ziclumeran (nex-z) and NTLA-2002, investigational in vivo CRISPR-based therapies with the potential to be the first single-dose treatment for ATTR amyloidosis and HAE respectively, will be presented.